Readily isolated bispecific antibodies with native immunoglobulin format

ABSTRACT

The invention relates to antigen-binding proteins or antibodies having heterodimers of heavy chains, i.e., two immunoglobulin heavy chains that differ by at least one or two amino acid(s) that allows for isolation of the antigen-binding protein based on a differential affinity of an immunoglobulin heavy chain and a modified/mutated immunoglobulin heavy chain toward an affinity reagent. The invention also relates antigen-binding proteins, including bispecific antibodies, having IgG CH1 regions with different affinities with respect to affinity reagent(s) that allows rapid isolation by differential binding of the IgG regions to the affinity reagent(s).

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/798,485, filed Mar. 13, 2013, which claims the benefit ofU.S. Provisional Application No. 61/610,141, filed Mar. 13, 2012. Thecontents of these applications are hereby incorporated by reference intheir entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The contents of the text file named “NOVI-027001USSeqListing.txt,” whichwas created on Nov. 30, 2015 and is 17 KB in size, are herebyincorporated by reference in their entirety.

FIELD OF INVENTION

The invention concerns antigen-binding proteins or antibodies havingheterodimers of heavy chains, i.e., two immunoglobulin heavy chains thatdiffer by at least one amino acid that allows for the isolation of theantigen-binding protein based on a differential affinity of animmunoglobulin heavy chain and a modified or mutated immunoglobulinheavy chain toward an affinity reagent. The invention also concernsantigen-binding proteins or antibodies having heterodimers of heavychains, i.e., two immunoglobulin heavy chains that differ by at leasttwo amino acids that allow for the isolation of the antigen-bindingprotein based on a differential affinity of an immunoglobulin heavychain and a modified or mutated immunoglobulin heavy chain toward anaffinity reagent. The invention also concerns antigen-binding proteins(including bispecific antibodies) that have IgG CH1 regions withdifferent affinities with respect to affinity reagent that allows rapidisolation by differential binding of the IgG regions to this affinityreagent.

BACKGROUND OF THE INVENTION

Antibodies are multifunctional molecules carrying a unique bindingspecificity for a target antigen or multiple targets and having thecapacity to interact with the immune system via mechanisms that areantigen-independent. Many currently used biological therapeutics forcancer are monoclonal antibodies directed against antigens that aretypically overexpressed on the targeted cancer cell. When suchantibodies bind tumor cells, they may trigger antibody-dependentcellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).Unfortunately, cancerous cells often develop mechanisms to suppressthese normal immune responses. In addition, targeting or neutralizing asingle protein is not always sufficient to achieve efficacy in certaindiseases which limits the therapeutic use of monoclonal antibodies. Itis increasingly clear that in a number of indications neutralizing onecomponent of a biological system is not sufficient to achieve efficacy.

Accordingly, there remains a need for a bispecific antibody format, inparticular for therapeutic applications, that minimizes some or all ofthe disadvantages mentioned above.

SUMMARY OF THE INVENTION

The invention is based at least in part on employing two immunoglobulinCH1 heavy chain constant domain sequences that differ by at least oneamino acid in a bispecific antigen-binding protein and form aheterodimer. The amino acid difference results in an improved ability toquickly and effectively isolate the heteromeric protein from homodimers,because the difference results in a differential ability of the CH1domain sequences to bind the CaptureSelect® IgG-CH1 (BAC BV) affinityreagent. In one aspect, an antigen-binding protein is provided,comprising a first and a second polypeptide, the first polypeptidecomprising, from N-terminal to C-terminal, a first antigen-bindingregion that selectively binds a first antigen, followed by a constantregion that comprises a first CH1 region of a human IgG selected fromIgG1 (SEQ ID NO: 1), IgG2 (SEQ ID NO: 2), IgG3 (SEQ ID NO: 3), IgG4 (SEQID NO: 4), and a combination thereof, and, a second polypeptidecomprising, from N-terminal to C-terminal, a second antigen-bindingregion that selectively binds a second antigen, followed by a constantregion that comprises a second CH1 region of a human IgG selected fromIgG1, IgG2, IgG3, IgG4, and a combination thereof, wherein the secondCH1 region comprises a modification that reduces or eliminates bindingof the second CH1 domain to the CaptureSelect® IgG-CH1 affinity reagent.

The invention is also based at least in part on employing twoimmunoglobulin CH1 heavy chain constant domain sequences that differ byat least two amino acids in a bispecific antigen-binding protein andform a heterodimer. The two amino acid difference results in an improvedability to quickly and effectively isolate the heteromeric protein fromhomodimers, because the difference results in a differential ability ofthe CH1 domain sequences to bind the CaptureSelect® IgG-CH1 (BAC BV)affinity reagent. In one aspect, an antigen-binding protein is provided,comprising a first and a second polypeptide, the first polypeptidecomprising, from N-terminal to C-terminal, a first antigen-bindingregion that selectively binds a first antigen, followed by a constantregion that comprises a first CH1 region of a human IgG selected fromIgG1 (SEQ ID NO: 1), IgG2 (SEQ ID NO: 2), IgG3 (SEQ ID NO: 3), IgG4 (SEQID NO: 4), and a combination thereof, and, a second polypeptidecomprising, from N-terminal to C-terminal, a second antigen-bindingregion that selectively binds a second antigen, followed by a constantregion that comprises a second CH1 region of a human IgG selected fromIgG1, IgG2, IgG3, IgG4, and a combination thereof, wherein the secondCH1 region comprises a modification that reduces or eliminates bindingof the second CH1 domain to the CaptureSelect® IgG-CH1 affinity reagent.

In one embodiment, the second CH1 region comprises mutations modifyingresidues S40 and T47, according to the IMGT exon numbering system(IMGT®, the international ImMunoGeneTics information System®).

In some embodiments, the second CH1 region comprises the S40T and T47Smodifications. As used herein, an “S40T” mutation is one in which thewild-type residue, serine, at position 40 is replaced with a threonine(i.e., S→T mutation at residue 40). Likewise, as used herein, a “T47S”mutation is one in which the wild-type residue, threonine, at position40 is replaced with a serine (i.e., T→S mutation at residue 47).

In specific embodiments, the second CH1 region is selected from SEQ IDNO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17.

In one embodiment, the second CH1 region is or is derived from amodified human IgG1 (SEQ ID NO: 14).

In one embodiment, the second CH1 region is or is derived from amodified human IgG2 (SEQ ID NO: 15).

In one embodiment, the second CH1 region is from a modified human IgG3(SEQ ID NO: 16).

In one embodiment, the second CH1 region is from a modified human IgG4(SEQ ID NO: 17).

In one embodiment, the CH1 domain is a chimeric domain that comprisessequences of two or more of human IgG1, human IgG2, human IgG3 and humanIgG4.

In one embodiment, the CH1 domain is from human IgG1, human IgG2, humanIgG3, or human IgG4, and the antigen-binding protein further comprises aCH2 domain and a CH3 domain, wherein the CH2 domain and the CH3 domainare independently selected from the group consisting of a human IgG1 CH2or CH3 domain, a human IgG2 CH2 or CH3 domain, a human IgG3 CH2 or CH3domain, a human IgG4 CH2 or CH3 domain.

In one embodiment, the antigen-binding protein further comprises animmunoglobulin light chain.

In other embodiment the immunoglobulin light chain is selected from ahuman lambda and a human kappa light chain.

In one embodiment, the first and the second antigen-binding regions eachcomprise at least one complementarity determining region (CDR). Inanother embodiment, the first and the second antigen-binding regionseach comprise at least two CDRs. In another embodiment, the first andthe second antigen-binding regions each comprise each comprise threeCDRs. In a specific embodiment, the CDRs are from an immunoglobulinheavy chain. In another specific embodiment, the heavy chain is a humanheavy chain.

In one embodiment, the first antigen-binding region comprises a firstimmunoglobulin heavy chain variable domain, and the secondantigen-binding region comprises a second immunoglobulin heavy chainvariable domain.

In one embodiment, the first and the second immunoglobulin heavy chainvariable domains independently comprise a human CDR, a mouse CDR, a ratCDR, a rabbit CDR, a monkey CDR, an ape CDR, a synthetic CDR, and/or ahumanized CDR. In one embodiment, the CDR is human and is somaticallymutated.

In one embodiment, the first and the second immunoglobulin heavy chainvariable domain comprise a human framework region (FR). In oneembodiment, the human FR is a somatically mutated human FR.

In one embodiment, the first and/or the second antigen-binding regionsare obtained by screening a phage library comprising antibody variableregions for reactivity toward an antigen of interest.

In another embodiment, the first and/or the second antigen-bindingregions are obtained by immunizing a non-human animal such as a mouse, arat, a rabbit, a monkey, or an ape with an antigen of interest andidentifying an antibody variable region nucleic acid sequence encodingvariable region specific for the antigen of interest.

In another specific embodiment, one or more human immunoglobulinvariable region genes are present in the non-human animalextrachromosomally, as a replacement at an endogenous immunoglobulinlocus, or as a transgene randomly integrated into the genome of thenon-human animal. In one embodiment, the first and/or the secondantigen-binding regions are obtained from a hybridoma or a quadroma, inanother embodiment from screening immune cells of an immunized non-humananimal using cell sorting.

In one embodiment, the antigen-binding protein is a bispecific antibody.In one embodiment, the bispecific antibody is a fully human bispecificantibody and has an affinity for each epitope, independently, in themicromolar, nanomolar, or picomolar range.

In one embodiment, the antigen-binding protein is non-immunogenic orsubstantially non-immunogenic in a human. In a specific embodiment, theantigen-binding protein lacks a non-native human T-cell epitope. In oneembodiment, the modification of the CH1 region is non-immunogenic orsubstantially non-immunogenic in a human.

In one embodiment, the antigen-binding protein comprises a heavy chain,wherein the heavy chain is non-immunogenic or substantiallynon-immunogenic in a human.

In one embodiment, the heavy chain has an amino acid sequence that doesnot contain a non-native T-cell epitope. In one embodiment, the heavychain comprises an amino acid sequence whose proteolysis cannot form anamino acid sequence of about 9 amino acids that is immunogenic in ahuman. In a specific embodiment, the human is a human being treated withthe antigen-binding protein. In one embodiment, the heavy chaincomprises an amino acid sequence whose proteolysis cannot form an aminoacid sequence of about 13 to about 17 amino acids that is immunogenic ina human. In a specific embodiment, the human is a human being treatedwith the antigen-binding protein.

In one aspect, the invention provides isolated multispecific antibodieshaving more than one antigen binding specificity, wherein themultispecific antibody include at least (i) a first polypeptidecomprising a first variable region that binds a first epitope and animmunoglobulin constant region that comprises a first CH1 region of ahuman IgG selected from IgG1, IgG2, IgG3 and IgG4, and (ii) a secondpolypeptide comprising a second region that binds a second epitope andan immunoglobulin constant region that comprises a second CH1 region ofa human IgG selected from IgG1, IgG2, IgG3 and IgG4, wherein the firstand second epitopes are different epitopes, and wherein at least one ofthe first and second CH1 regions comprises a modification that reducesor eliminates binding of the second CH1 domain to the CaptureSelect®IgG-CH1 affinity reagent, or any affinity reagent targeting the humanIgGi, IgG2, IgG3 and IgG4 CH1 domain.

The invention also provides heterodimeric bispecific antigen-bindingproteins that include (a) a first polypeptide comprising, fromN-terminal to C-terminal a first epitope-binding region that selectivelybinds a first epitope and an immunoglobulin constant region thatcomprises a first CH1 region of a human IgG selected from IgG1, IgG2,IgG3 and IgG4; and, b) a second polypeptide comprising, from N-terminalto C-terminal, a second epitope-binding region that selectively binds asecond epitope and an immunoglobulin constant region that comprises asecond CH1 region of a human IgG selected from IgG1, IgG2, IgG3 andIgG4, wherein the second CH1 region comprises a modification thatreduces or eliminates binding of the second CH1 domain to theCaptureSelect® IgG-CH1 affinity reagent, or any affinity reagenttargeting the human IgG1, IgG2, IgG3 and IgG4 CH1 domain.

In some embodiments of the multispecific antibodies, e.g., heterodimericbispecific antigen-binding proteins, the first polypeptide and thesecond polypeptide comprise human IgG heavy chains or are derived fromhuman IgG heavy chains. In some embodiments, the multispecificantibodies, e.g., heterodimeric bispecific antigen-binding proteins,also include an immunoglobulin light chain. In some embodiments of themultispecific antibodies, e.g., heterodimeric bispecific antigen-bindingproteins, the immunoglobulin light chain is a human immunoglobulin lightchain or is derived from a human immunoglobulin light chain. In someembodiments of the multispecific antibodies, e.g., heterodimericbispecific antigen-binding proteins, the first and the secondpolypeptides each are human IgG1 heavy chains or are derived from humanIgG1 heavy chains. In some embodiments of the multispecific antibodies,e.g., heterodimeric bispecific antigen-binding proteins, the second CH1region comprises the modification. In some embodiments of themultispecific antibodies, e.g., heterodimeric bispecific antigen-bindingproteins, the modification in the second CH1 domain include a mutationmodifying S40 in the IMGT exon numbering system, a mutation modifyingT47 in the IMGT exon numbering system or a combination thereof. In someembodiments of the multispecific antibodies, e.g., heterodimericbispecific antigen-binding proteins, the modification in the second CH1domain comprises an S40T mutation in the IMGT exon numbering system, aT47S mutation in the IMGT exon numbering system or a combinationthereof. In some embodiments of the multispecific antibodies, e.g.,heterodimeric bispecific antigen-binding proteins, the first CH1 domainof the bispecific antibody, the second CH1 domain or both the first andsecond CH1 domains are non-immunogenic or substantially non-immunogenicin a human.

In one aspect, a method for making a bispecific antibody is provided,comprising: obtaining a nucleic acid sequence encoding a firstimmunoglobulin heavy chain comprising a first variable domain thatrecognizes a first epitope, wherein the first immunoglobulin heavy chaincomprises an IgG1, IgG2, IgG3 or IgG4 isotype constant domain; obtaininga second nucleic acid sequence encoding a second immunoglobulin heavychain comprising a second variable domain that recognizes a secondepitope, wherein the second immunoglobulin heavy chain comprises anIgG1, IgG2, IgG3 or IgG4 isotype constant domain, or a chimeric isotypeconstant domain thereof, that comprises a modification in its CH1 domainthat eradicates or reduces binding to the CaptureSelect® IgG-CH1affinity reagent; obtaining a third nucleic acid sequence encoding animmunoglobulin a light chain that pairs with the first and the secondimmunoglobulin heavy chain; introducing the first, second, and thirdnucleic acid sequences into a mammalian cell; allowing the cell toexpress an immunoglobulin, and isolating the immunoglobulin using theCaptureSelect® IgG-CH1 affinity reagent.

In one embodiment, the cell is selected from a CHO, COS, 293, HeLa, anda retinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6™cell).

In one aspect, a method for making a bispecific antibody is provided,comprising a step of isolating from a disrupted cell or a mixture ofantibodies a bispecific antibody having differentially modified IgGi,IgG2, IgG3 or IgG4 CH1 domains, wherein the differentially modified CH1domains are non-immunogenic or substantially non-immunogenic in a human,and wherein the modification results in a bispecific antibody withheterodimeric heavy chains whose monomers have a differential affinityfor an affinity reagent, and the bispecific antibody is isolated fromthe disrupted cell or the mixture using an affinity reagent.

In one embodiment, the heterodimeric bispecific antibody can bepreferentially purified at specific pH range and salt concentration. Inthis embodiment, the heterodimeric bispecific antibody is composed oftwo different heavy chains, one modified at positions 40 and 47 (IMGT®numbering), or at positions 40, 45 and 47 (IMGT® numbering), on its CH1domain; and the other one lacks modification at positions 40 and 47(IMGT® numbering), or at positions 40, 45 and 47 (IMGT® numbering), onits CH1 domain.

In one aspect, the invention provides methods for producingmultispecific antibodies, e.g., heterodimeric bispecific antigen-bindingproteins, by (a) obtaining a nucleic acid sequence encoding a firstimmunoglobulin heavy chain comprising a first variable domain thatrecognizes a first epitope, wherein the first immunoglobulin heavy chaincomprises an IgG1, IgG2, IgG3 or IgG4 isotype constant domain; (b)obtaining a second nucleic acid sequence encoding a secondimmunoglobulin heavy chain comprising a second variable domain thatrecognizes a second epitope, wherein the second immunoglobulin heavychain comprises an IgG1, IgG2, IgG3 or IgG4 isotype constant domain thatcomprises a modification in its CH1 domain that eradicates or reducesbinding to CaptureSelect® IgG-CH1 affinity reagent, or any affinityreagent targeting the human IgG1, IgG2, IgG3 and IgG4 CH1 domain; (c)obtaining a third nucleic acid sequence encoding an immunoglobulin lightchain that pairs with the first and the second immunoglobulin heavychain; (d) introducing the first, second, and third nucleic acidsequences into a mammalian cell; (e) allowing the cell to express abispecific antibody; and (f) isolating the bispecific antibody based onthe ability of the bispecific antibody to bind the CaptureSelect®IgG-CH1 affinity reagent, or any affinity reagent targeting the humanIgG1, IgG2, IgG3 and IgG4 CH1 domain.

In some embodiments, the modification in the second CH1 domain comprisesan S40T mutation in the IMGT exon numbering system, a T47S mutation inthe IMGT exon numbering system or a combination thereof. In someembodiments, the first CH1 domain of the bispecific antibody, the secondCH1 domain or both the first and second CH1 domains are non-immunogenicor substantially non-immunogenic in a human. In some embodiments, thebispecific antibody is isolated on a solid support comprising aCaptureSelect® IgG-CH1 affinity reagent, or any affinity reagenttargeting the human IgG1, IgG2, IgG3 and IgG4 CH1 domain. In someembodiments, the solid support comprises a CaptureSelect® IgG-CH1affinity column, or any affinity reagent targeting the human IgG1, IgG2,IgG3 and IgG4 CH1 domain, and the bispecific antibody is isolatedemploying a pH gradient. In some embodiments, the pH gradient is a stepgradient comprising one or more pH steps between pH 3 and pH 5.

In one aspect, the invention provides methods for isolating a bispecificantibody by isolating from a disrupted cell or a mixture of antibodies abispecific antibody having differentially modified IgG1, IgG2, IgG3 orIgG4 CH1 domains, wherein the differentially modified CH1 domains arenon-immunogenic or substantially non-immunogenic in a human, and whereinthe modification results in a bispecific antibody with a heterodimericheavy chain constant region whose monomers have a differential affinityfor the CaptureSelect® IgG-CH1 affinity reagent, or any affinity reagenttargeting the human IgG1, IgG2, IgG3 and IgG4 CH1 domain, and thebispecific antibody is isolated from the disrupted cell or the mixturebased on its affinity for the CaptureSelect® IgG-CH1 affinity reagent,or any affinity reagent targeting the human IgG1, IgG2, IgG3 and IgG4CH1 domain.

In some embodiments, one monomer of the heterodimeric heavy chainconstant region is a human IgG1 and the other monomer of theheterodimeric heavy chain constant region is a modified human IgG1comprising a modification selected from the group consisting an S40Tmutation in the IMGT exon numbering system, a T47S mutation in the IMGTexon numbering system or a combination thereof. In some embodiments, thefirst immunoglobulin heavy chain comprises a mutation or modificationaltering its binding properties to an affinity chromatography resin. Insome embodiments, the first immunoglobulin heavy chain comprises amutation altering its binding properties to Protein A. In someembodiments, the first immunoglobulin heavy chain comprises a H435Rmutation altering its binding properties to Protein A.

Any of the embodiments and aspects described herein can be used inconjunction with one another, unless otherwise indicated or apparentfrom the context. Other embodiments will become apparent to thoseskilled in the art from a review of the ensuing description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a table showing the specificity of the CaptureSelect® IgG-CH1affinity reagent for known purified antibodies of different species anddifferent isotypes.

FIG. 2 is an illustration depicting the sequence alignment of the CH1domains of the following species and isotypes: human IGHG1 (H-IGHG1, SEQID NO: 1), human IGHG2 (H-IGHG2, SEQ ID NO: 2), human IGHG3 (H-IGHG3,SEQ ID NO: 3), human IGHG4 (H-IGHG4, SEQ ID NO:4), hamster IGHG(Ha-IGHG, SEQ ID NO: 5), mouse IGHG1 (M-IGHG1, SEQ ID NO: 6), mouseIGHG2A (M-IGHG2A, SEQ ID NO: 7), mouse IGHG2B (M-IGHG2B, SEQ ID NO: 8),mouse IGHG2C (M-IGHG2C, SEQ ID NO: 9), mouse IGHG3 (M-IGHG3, SEQ ID NO10), rat IGHG1 (R-IGHG1, SEQ ID NO: 11), rat IGHG2A (R-IGHG2A, SEQ IDNO: 12), rat IGHG2B (R-IGHG2B, SEQ ID NO: 13), and rat IGHG2C (R-IGHG2C,SEQ ID NO: 14).

FIG. 3 is an illustration depicting an example of a bispecific antibodyhaving wild-type CH1 domain with a serine residue at position 40 (40S)and a threonine residue at position 47 (47T).

FIG. 4 is a table depicting the CH1 domain mutations tested in theexamples provided below.

FIG. 5 is an illustration depicting the sequence alignment of theH-IGHG1 (SEQ ID NO: 1) CH1 domain and the H-IGHG1 M2 (SEQ ID NO: 15) CH1domain, where the H-IGHG1 M2 mutant is a variant of the H-IGHG1 sequencehaving a threonine at position 40 (also referred to herein as an S40Tmutation) and a serine at position 47 (also referred to herein as a T47Smutation).

FIG. 6 is a graph depicting the amount of purified antibodies obtainedafter purification on either a Protein A affinity column or aCaptureSelect® IgG-CH1 affinity column.

FIG. 7 is a graph depicting the percentage of purified antibody recoveryusing a Protein A affinity column as compared to the purified antibodyrecovery using a CaptureSelect® IgG-CH1 affinity column.

FIG. 8 is an illustration depicting the sequence alignment of theH-IGHG1 (SEQ ID NO: 1) CH1 domain and the H-IGHG1 M2 (SEQ ID NO: 15) CH1domain; the H-IGHG2 (SEQ ID NO: 2) CH1 domain and the H-IGHG2 M2 (SEQ IDNO: 16) CH1 domain, where the H-IGHG2 M2 mutant is a variant of theH-IGHG2 sequence having a threonine at position 40 (also referred toherein as an S40T mutation) and a serine at position 47 (also referredto herein as a T47S mutation); the H-IGHG3 (SEQ ID NO: 3) CH1 domain andthe H-IGHG3 M2 (SEQ ID NO: 17) CH1 domain, where the H-IGHG3 M2 mutantis a variant of the H-IGHG3 sequence having a threonine at position 40(also referred to herein as an S40T mutation) and a serine at position47 (also referred to herein as a T47S mutation); and the H-IGHG4 (SEQ IDNO: 4) CH1 domain and the H-IGHG4 M2 (SEQ ID NO: 18) CH1 domain, wherethe H-IGHG4 M2 mutant is a variant of the H-IGHG42 sequence having athreonine at position 40 (also referred to herein as an S40T mutation)and a serine at position 47 (also referred to herein as a T47Smutation).

FIG. 9 is an illustration depicting a possible bispecific antibodyformat purified by two affinity steps. This antibody has one commonlight chain and two different heavy chains. Mutations have beenintroduced in these two different heavy chains. One heavy chains hasS40T and T47S mutations in its CH1 domain which abrogate binding toCaptureSelect® IgG-CH1 affinity reagent, while the other chain has H435Rmutation in its CH3 domain which abrogates binding to protein A. Thepositions that were mutated are indicated.

FIG. 10 is an illustration depicting the asymmetric purificationstrategy used to isolate bispecific antibody from the mixture ofantibodies generated upon expression in single cell of two heavy chainsand one light chain. The monospecific antibody carrying the H435R onboth heavy chains does not bind to the Protein A resin and is lost inthe flow-through. The second monospecific antibody carrying the S40T andT47S mutations on both heavy chains does not bind to the CaptureSelect®IgG-CH1 affinity reagent and is lost in the flow-through. The bispecificantibody is recovered in the final elution step.

FIG. 11 is a graph depicting antibody binding results against CD3obtained by ELISA. The ELISA format is described by a cartoon in theright panel.

FIG. 12 is a graph depicting antibody binding results against IL-17obtained by ELISA. The ELISA format is described by a cartoon in theright panel.

FIG. 13 is a graph depicting antibody co-engagement results against bothCD3 and IL-17 obtained by ELISA. The ELISA format is described by acartoon in the right panel.

FIG. 14 is a graph depicting sensorgrams obtained on a Biacore 2000instrument for the interaction between the biotinylated ligand ofCaptureSelect® IgG-CH1 immobilized on a streptavidin coated CM5 chip,with different IgG bearing unmodified or modified CH1 domains. Themutated hIgG1 carry the mutations described in FIG. 4.

DETAILED DESCRIPTION

In order to overcome the limitations of monoclonal and monovalentantibody therapeutics that can only target a single antigen or toovercome the limitations of combinations of monovalent antibodytherapeutics, intense efforts have aimed at multiple antigen targetingusing bispecific antibody formats. Bispecific antibodies areadvantageous as they allow for multiple targeting, they increasetherapeutic potential, they address redundancy of biological systems,and they provide novel mechanisms of action through abilities such asretargeting and/or increased specificity. As validated singletherapeutic targets become more and more exhausted, combinations allowedby bispecific antibodies provide a new and expansive universe of targetsfor therapeutic agents and applications.

In recent years, efforts have been underway to develop antibody liketherapeutics that have more than one antigen binding specificity, e.g.,bispecific antibodies. In the case of cancer therapies, multi-specificformats could allow the possibility of using, e.g., one specificity totarget the molecule to a tumor cell antigen, the other specificity totrigger a response that is not normally available to the immune system.Bispecific antibodies may also find use as surrogate ligands fortwo-component heterodimeric receptor systems that are normally activatedby their natural ligand when it binds to and brings together bothcomponents.

Numerous formats have been developed in the art to address therapeuticopportunities afforded by molecules with multiple binding specificities.Ideally, such molecules should be well-behaved proteins that are easy toproduce and purify, and possess favorable in vivo properties. e.g.,pharmacokinetics appropriate for an intended purpose, minimalimmunogenicity, and, if desirable, effector functions of conventionalantibodies.

The most straightforward way of producing a bispecific antibody(expressing two distinct antibodies in a single cell) gives rise tomultiple species, because the respective heavy chains form both homo-and heterodimers, but only the heterodimers are desired. Also, the lightand heavy chains may pair inappropriately. Several examples of formatsthat attempt to address these problems in different ways are describedbelow.

One format, used for Bispecific T cell Engager (BiTE) molecules (see,e.g., Wolf, E. et al. (2005) Drug Discovery Today 10:1237-1244)), isbased on single chain variable fragment (scFv) modules. An scFv consistsof an antibody's light and heavy chain variable regions fused via aflexible linker, which generally can fold appropriately and so that theregions can bind the cognate antigen. A BITE concatenates two scFv's ofdifferent specificities in tandem on a single chain. This configurationprecludes the production of molecules with two copies of the same heavychain variable region. In addition, the linker configuration is designedto ensure correct pairing of the respective light and heavy chains.

The BITE format has several disadvantages. First, scFv molecules arenotorious for their tendency to aggregate. And although theimmunogenicity of scFv linkers is reputedly low, the possibility ofgenerating antibodies against a BITE cannot be ruled out. The absence ofan Fc portion in the BITE format also makes its serum half-life veryshort, and this necessitates the complication of frequent repeatedadministrations or continuous infusion via a pump. Finally, the absenceof an Fc also implies the absence of Fc-mediated effector functions,which may be beneficial in some circumstances.

A second format is a hybrid of a mouse and a rat monoclonal antibody,and relies on a modification of conventional Protein A affinitychromatography (see, e.g., Lindhofer, H. et al. (1995) J. Immunol.155:219-225)). In this format, a mouse IgG2a and a rat IgG2b antibodyare produced together in the same cell (e.g., either as a quadromafusion of two hybridomas, or in engineered CHO cells). Because the lightchains of each antibody associate preferentially with the heavy chainsof their cognate species, only three distinct species of antibody can beassembled: the two parental antibodies, and a heterodimer of the twoantibodies comprising one heavy/light chain pair of each, associatingvia their Fc portions. The desired heterodimer can be easily purifiedfrom this mixture because its binding properties to Protein A aredifferent from those of the parental antibodies: rat IgG2b does not bindto protein A, whereas the mouse IgG2a does. Consequently, the mouse-ratheterodimer binds to Protein A but elutes at a higher pH than the mouseIgG2a homodimer, and this makes selective purification of the bispecificheterodimer possible. As with the BITE format, this hybrid format hastwo monovalent antigen binding sites.

The disadvantage of the mouse/rat hybrid is that because it isnon-human, it is likely to provoke an immune response in the patient,which could have deleterious side effects, and/or neutralize thetherapeutic.

Based on the concept described above, i.e., differential binding of aheterodimeric molecule to Protein A, other formats relying on themodification of the Fc region responsible for binding to protein A havebeen described in US Patent Application Publication No. 20100331527A1. Alimitation of engineering the Fc region of an antibody is that themutations introduced might affect the interaction with Fc receptors and,therefore, Fc mediated functions. In particular, the FcRn interaction iscrucial for long half-life of antibodies and this interaction site islocated close to the Protein A binding site.

Another format, referred to as “knobs-into-holes” has been discussed inthe prior art as potentially useful for the production of bispecificantibodies (U.S. Pat. No. 7,183,076). In this strategy, the Fc portionsof two antibodies are engineered to give one a protruding “knob”, andthe other a complementary “hole.” When produced in the same cell, theheavy chains are said to preferentially form heterodimers rather thanhomodimers, by association of the engineered “knobs” with the engineered“holes.” Issues of correct light-heavy chain pairing are addressed bychoosing antibodies that have different specificities but employidentical light chains.

The disadvantage of this format is that the “knobs-into-holes” strategycan result in production of a significant amount of undesirablehomodimers, thus necessitating further purification steps. Thisdifficulty is exacerbated by the fact that the contaminating species arenearly identical to the desired species in many of their properties. Theengineered forms may also potentially be immunogenic, because themutations producing the “knobs” and “holes” introduce foreign sequences.

The approach to generate bispecific antibodies described hereinovercomes the disadvantages of other it does not involve mutagenesis ofthe Fc region but instead relies on CH1 domain modifications which alterits binding capacity to a CH1 specific affinity chromatography media.The CH1 region of antibodies is not known to be involved in interactionswith receptors or other proteins and thus the effector functions andpharmacokinetic properties of the bispecific format of the inventionremain unaltered. An additional benefit of some of the mutations of theCH1 domain described herein is that even very conservative changes(i.e., serine to threonine and threonine to serine) can abolish bindingto the CH1 specific chromatography media. Although, these conservativemutations represent preferred changes, those ordinary skilled in the artwill appreciate that less conservative mutations can also be applied forthis approach.

The present invention allows for the purification of the bispecificantibody species that contains only one unmodified CH1 domain frommonospecific antibodies. This can be achieved by co-expressing in asingle cell two different antibody heavy chains and one antibody lightchain. The two heavy chains, when paired with the common light chainmediate specific binding against two different antigens. One of the twoheavy chains contains a modified CH1 domain that prevents its binding tothe CH1 specific chromatography media. The co-expression of the threechains leads to the production of a mixture of three antibodies: ahomodimeric monospecific antibody bearing two unmodified CH1 domains, ahomodimeric monospecific antibody bearing two modified CH1 domains and aheterodimeric bispecific antibody bearing one modified CH1 domain andone unmodified CH1 domain. The differential properties of the threedifferent molecules can exploited to efficiently purify the bispecificantibody from the monospecific ones.

The modifications of the CH1 domain described herein can be combinedwith other mutations or modification of a portion of an antibody, suchas the Fc domain. Such combinations enable a two-step asymmetricpurification approach that further facilitates isolation of thebispecific molecule.

EXAMPLES

The following examples are provided to describe to those of ordinaryskill in the art how to make and use methods and compositions of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isaverage molecular weight, temperature is in degrees Centigrade, andpressure is at or near atmospheric.

Example 1. The CaptureSelect® IgG-CH1 Affinity Reagent SpecificallyBinds to Human and Hamster IgGs

Binding experiments were conducted with known purified antibodies ofdifferent species and different isotypes to test the specificity of theCaptureSelect® IgG-CH1 affinity reagent. These experiments revealed thatonly human and hamster IgGs could be retained on the CaptureSelect®IgG-CH1 affinity resin while mouse and rat IgGs of different isotypescouldn't bind to this affinity reagent (FIG. 1).

Example 2. Identification of Sequence Determinants Responsible of theIgG Binding to the CaptureSelect® IgG-CH1 Affinity Reagent

Sequence alignment of human (IGHG1 SEQ ID NO: 1, IGHG2 SEQ ID NO: 2,IGHG3 SEQ ID NO: 3, IGHG4 SEQ ID NO:4), hamster (IGHG SEQ ID NO: 5),mouse (IGHG1 SEQ ID NO: 6, IGHG2A SEQ ID NO: 7, IGHG2B SEQ ID NO: 8,IGHG2C SEQ ID NO: 9, IGHG3 SEQ ID NO 10) and rat (IGHG1 SEQ ID NO: 11,IGHG2A SEQ ID NO: 12, IGHG2B SEQ ID NO: 13, IGHG2C SEQ ID NO: 14)isotypes was carried out (FIG. 2). Two residues conserved in human andhamster CH1 but not in mouse and rat sequences were identified: 40S and47T. Their exposures to the solvent were also determined in antibodystructure, indicating that these residues are highly accessible. Anotherresidue 45A is located in the vicinity of 40S and 47T, is well exposedto the solvent and is partially conserved in the different species (FIG.3).

Example 3. Modification of Human CH1 Domain to Alter Binding to CH1Affinity Chromatography Media

Modification of human IGHG1 were introduced in the CH1 domain bysite-directed mutagenesis as single (S40T or T47S) double (S40T, T47S)or triple (S40T, A45S, T47S) mutations and were called M2ST, M2TS, M2and M3, respectively (FIGS. 4, 5 and 8). These different mutants werefurther expressed in 293 cells using standard mammalian celltransfection procedures and purified from the supernatant as describedbelow.

Protein A and CaptureSelect® IgG-CH1 purification of differentiallymodified IgGl: Differentially modified monomeric IgG1 (ref e.g. WT,M2ST, M2TS, M2 and M3) were expressed in 293 cells. Cell supernatantswere harvested and divided in two. Antibodies were then purified eitheron Protein A or CaptureSelect® IgG-CH1 affinity columns. The amount ofpurified antibodies obtained after purification were determined (FIG.6). Percentage of antibody recovery between Protein A and CaptureSelect®IgG-CH1 affinity columns were also calculated (FIG. 7). This percentageindicated that single mutations M2ST and M2TS lowered the antibodybinding to the CaptureSelect® IgG-CH1 affinity column but didn'tabrogate it. Double and triple mutants M2 and M3 recoveries onCaptureSelect® IgG-CH1 affinity column were extremely low indicatingthat the S40T, T47S double mutation was sufficient to abrogate IGHG1binding to this affinity reagent.

Example 4. Generation and Purification of an Anti-IL17 x Anti-CD3Bispecific Antibody

It was found that two known antibodies of human IgG1 isotype, oneagainst IL-17 and one against protein CD3, had light chains thatdiffered by only one amino acid. Co-expression experiments revealed thatthe light chain of the anti-IL-17 antibody could be replaced with thelight chain from the anti-CD3 antibody and still maintain high affinitybinding to IL-17, thus making it feasible to produce a bispecificantibody using the anti-IL-17 heavy chain, the anti-CD3 heavy chain andthe same light chain. Accordingly, the heavy chain of the anti-IL-17antibody was modified to the M2 form (i.e., CH1 modifications S40T andT47S, by IMGT® exon numbering).

Moreover, to facilitate the purification of the bispecific antibody,H435R mutation was introduced in the CH3 domain of the heavy chain ofthe anti-CD3 antibody to disrupt binding to protein A affinity reagent(FIG. 9). Based on the paper of Natsume et al. (Cancer Res 2008;68:3863-3872, the contents of which are hereby incorporated by referencein their entirety) who engineered the C-terminal part of the CH3 domainof the IgG3-Fc region to introduce binding of IgG3 to protein A, theC-terminal residues of the CH3 domain of IgG3 were introduced in humanIgG1. Only two residues differ between IgG1 and IgG3 in this part of CH3domain, which corresponds to residues His435 and Tyr436 (which are Argand Phe residues in IgG3, respectively). After site-directedmutagenesis, H435R mutation proved to be sufficient to abrogate bindingto protein A. Therefore, the anti-CD3 and anti-IL-17 heavy chains wereco-expressed with the same light chain in 293 cells, and the bispecificantibody was purified from supernatant using asymmetric purification. Afirst purification was conducted with the protein A resin to capturebispecific antibody and anti-IL17 monospecific antibody. Then, a secondpurification with CaptureSelect® IgG-CH1 affinity column was used tospecifically capture bispecific antibody (FIG. 10). Finally, ELISAexperiments were conducted to determine the antibody binding againstCD3, IL-17 or co-engagement of both CD3 and IL-17. Only, the bispecificantibody obtained by asymmetrical purification was able to engage bothIL-17 and CD3 when compared to the monospecific antibodies (FIG. 11-13).

Example 5. Binding of Antibodies Containing Mutated CH1 Domains to theLigand of the CaptureSelect® IgG-CH1 Affinity Resin by Surface PlasmonResonance

The interaction of the IgG bearing modified and unmodified CH1 domainswas characterized using surface Plasmon resonance on Biacore 2000instrument. The biotinylated ligand of the CaptureSelect® IgG-CH1affinity resin (BAC BV) was immobilized on a streptavidin coated CM5chip (GE Healthcare). The M2, M3, M2ST and unmodified IgG1 were injectedon the surface and the sensorgram recorded (FIG. 14). A clearinteraction between the IgG containing an unmodified CH1 domain and theligand of the CaptureSelect® IgG-CH1 affinity resin can be observed,with a very slow off rate. In contrast, the double and triple mutationspresent in the M2 and M3 completely abolish this interaction. The singlemutation S40T leads to a partial inhibition of the interaction.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

1.-8. (canceled)
 9. A method for making a multispecific antibodycomprising: a) obtaining a nucleic acid sequence encoding a firstimmunoglobulin heavy chain comprising a first variable domain thatrecognizes a first epitope, wherein the first immunoglobulin heavy chaincomprises an IgG1, IgG2, IgG3 or IgG4 isotype constant domain; b)obtaining a second nucleic acid sequence encoding a secondimmunoglobulin heavy chain comprising a second variable domain thatrecognizes a second epitope, wherein the second immunoglobulin heavychain comprises an IgG1, IgG2, IgG3 or IgG4 isotype constant domain, ora chimeric isotype constant domain thereof, that comprises amodification in its CH1 domain that eradicates or reduces binding to theCaptureSelect® IgG-CH1 affinity reagent; c) obtaining a third nucleicacid sequence encoding an immunoglobulin a light chain that pairs withthe first and the second immunoglobulin heavy chain; d) introducing thefirst, second, and third nucleic acid sequences into a mammalian cell;e) allowing the cell to express an immunoglobulin; and f) isolating theimmunoglobulin using the CaptureSelect® IgG-CH1 affinity reagent, or anyaffinity reagent targeting the human IgG1, IgG2, IgG3 and IgG4 CHIdomain.
 10. The method of claim 9, wherein the cell is selected from aCHO, COS, 293, HeLa, and a retinal cell expressing a viral nucleic acidsequence (e.g., a PERC.6™ cell).
 11. The method of claim 9, wherein themethod comprises a step of isolating from a disrupted cell or a mixtureof antibodies a multispecific antibody having differentially modifiedIgG1, IgG2, IgG3 or IgG4 CH1 domains, wherein the differentiallymodified CH1 domains are non-immunogenic or substantiallynon-immunogenic in a human, and wherein the modification results in amultispecific antibody with heterodimeric heavy chains whose monomershave a differential affinity for an affinity reagent, and themultispecific antibody is isolated from the disrupted cell or themixture using an affinity reagent.
 12. The method of claim 9, whereinthe multispecific antibody can be preferentially purified at specific pHrange and salt concentration.
 13. The method of claim 9, wherein themultispecific antibody is composed of two different heavy chains, onemodified at positions 40 and 47 (IMGT® numbering), or at positions 40,45 and 47 (IMGT® numbering), on its CH1 domain; and the other one lacksmodification at positions 40 and 47 (IMGT® numbering), or at positions40, 45 and 47 (IMGT® numbering), on its CH1 domain.
 14. The method ofclaim 9, wherein the modification in the second CH1 domain comprises anS40T mutation in the IMGT exon numbering system, a T47S mutation in theIMGT exon numbering system or a combination thereof.
 15. The method ofclaim 14, wherein the first CH1 domain of the multispecific antibody,the second CH1 domain or both the first and second CH1 domains arenon-immunogenic or substantially non-immunogenic in a human.
 16. Themethod of claim 9, wherein the multispecific antibody is isolated on asolid support comprising a CaptureSelect® IgG-CH1 affinity reagent, orany affinity reagent targeting the human IgG1, IgG2, IgG3 and IgG4 CH1domain.
 17. The method of claim 16, wherein the solid support comprisesa CaptureSelect® IgG-CH1 affinity column, or any affinity reagenttargeting the human IgG1, IgG2, IgG3 and IgG4 CH1 domain, and themultispecific antibody is isolated employing a pH gradient.
 18. Themethod of claim 17, wherein the pH gradient is a step gradientcomprising one or more pH steps between pH 3 and pH
 5. 19. The method ofclaim 9, wherein the first immunoglobulin heavy chain comprises amutation or modification altering its binding properties to an affinitychromatography resin.
 20. The method of claim 19, wherein the firstimmunoglobulin heavy chain comprises a mutation altering its bindingproperties to Protein A.
 21. The method of claim 20, wherein the firstimmunoglobulin heavy chain comprises a H435R mutation altering itsbinding properties to Protein A.